Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca21. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter, N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3-5 min and declined to base line in 30min. The response was dose-dependent (EC50 ;100mm) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitor NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. Ng was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione, and H2O2 at concentrations less than 0.5mm. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca21 and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.